Cancer diagnosis method

ABSTRACT

The invention concerns a method for the diagnosis and/or the follow up of the evolution of cancer comprising the analysis of the RNA components of the telomerase enzyme present in the plasma or serum of the blood.

This application is a national phase application of International Application No. PCT/IB01/00852, having an International Filing Date of May 16, 2001 which application claims priority under the terms of the Paris Convention and 35 U.S.C. §§ 120 and 361 to European Application No. EP 000111370.3, filed May 26, 2000, the disclosures thereof being explicitly incorporated by reference herein.

The present invention relates to a method of diagnosis and/or follow up of the evolution of several types of cancer, for instance after a chemotherapy or after an operation.

It is known that diagnosis and follow up of the evolution of cancers are carried out, besides direct observation of the tumors, by biopsy analysis or in the case of blood malignancies by analysis of the bone marrow, which implies either a surgical intervention, or an invasive test such as a biopsy or a bone marrow aspiration. Now, in addition to the disagreeable or even dangerous aspect of such methods, it has been observed that they could moreover not be very precise. In the case of breast cancer, great efforts have been made to develop a detection test by mammography. Although several studies indicate that mass mammography may be a useful strategy to reduce breast cancer mortality, this method involves a certain number of disadvantages. Amongst these, a high rate of false positives, frequent false negatives and enormous public health costs should be underlined. Thus, when the benefits are weighed against these advantages, it is not surprising that this form of screening has engendered contentious debates over the last twenty years.

The aim of this invention consists therefore in providing a method of diagnosis and/or follow up of the evolution of several types of cancers which would be, on one hand more precise and reliable and, on the other hand easier to perform without implying an invasive test for the patient.

The method of diagnosis and/or follow up of the evolution of cancer, object of the invention aiming to reach the above cited goal, includes the analysis of the RNA component and the mRNA coding for the proteins of the ribonucleoprotein telomerase in the blood plasma or serum.

Telomerase is a ribonucleoprotein enzyme that synthesizes repeated telomeric sequences at chromosomal ends. The telomeres protect the chromosomal ends and at each cell division these telomeres are shortened. Telomerase uses in order to synthesise these telomeres one or two segments of its RNA components as template.

The activity of this enzyme has become an accepted indicator for the diagnosis and the prognosis of most malignant tumors. The expression of human telomerase RNA (hTR) or of the reverse transcriptase enzyme of the RNA telomerase (hTERT) or/of the associated protein (TEP1) has been measured during the progression of several types of tumors. This has enabled the establishment of a correlation between this expression (the amount of RNA) and telomerase activity. Most cancers and immortalized cell lines have a high telomerase activity that reflects a mechanism that escapes normal aging regulations.

Now, although RNA components and mRNA coding for telomerase are cellular components, it was observed that, surprisingly, these components could be also found in an extracellular form in plasma or serum.

Indeed the present inventors have shown the presence of hTR, hTERT and TEP1 in the plasma or serum of persons suffering especially from breast, ovarian, stomach or colon cancers, while these products have been shown to be absent in the blood of healthy persons. Contrarily to several deoxyribonucleic acid (DNA) markers that has already been found in plasma or serum, such as Ras gene or P53 mutations or with the numerous the microsatellite sequences found in the literature, the telomerase RNAs seem to be able to be used for every kind of tumors. It appears to be a general plasmatic or serum cancer marker. Moreover for most cancers, the rate of sera bearing telomerase RNA is more important than the rate obtained with other nucleic acid markers used up to now.

More precisely, the method of diagnosis according to the invention consists in extracting the RNA from the plasma or the serum of the blood, purifying it and amplifying it in order to establish the presence and if necessary the quantity of the product made by the reverse polymerase chain reaction (RT-PCR) representing components hTR, hTERT or TEP1, this in a comparative manner between the plasma or serum of a person suspected of malignancy and the plasma or serum of a healthy person.

The PCR amplification products of the RNA components transcribed into DNA by the RT-PCR are detected and if necessary quantified using for instance gel coloration methods. This analysis can be performed also differently on other gels or without gel by a radioactive immunological technique (RIA), by ELISA (enzyme linked immunosorbant assay) or by a microchip test (gene array). A quantitative amplification on a Taq Man cycler (Perkin Elmer Biosystems) or a capillary Light Cycler (Hofmann La Roche) improves the precision of hTR. HTERT and TEP 1 detection.

Similarly, any technique of extraction of purification and of amplification of the RNA in the plasma or the serum may be used.

The present invention will now be illustrated in a non-limitative manner by the following Example related to breast cancer diagnosis. However, it must be stressed that the scope of the present invention is no way limited to the diagnosis of this type of cancer. Preliminary data have indeed also shown the presence of hTR, hTERT and TEP1 in the serum of patients suffering especially of ovarian or gastrointestinal cancers. On the basis of these results, it can be therefore concluded that hTR, hTERT and TEP1 in plasma or serum may constitute general markers for numerous types of cancers.

EXAMPLE

Diagnosis of breast cancer by the detection of hTR, hTERT or TEP1 in the plasma or serum of the blood.

Blood samples (2 to 3 ml) were collected prior to surgery on 18 patients bearing small malignant breast tumors (T1 or T2) still differentiated (G1 or G2) and without cancerous nodules nor metastases. After clotting, tubes were centrifuged at 900 g for 15 minutes at room temperature and serum collected. This was followed by a second 15 minutes centrifugation at 900 g to remove any cellular debris. Serum samples were stored at −70° C. until use.

RNA was extracted using a commercially available kit (SV Total RNA Isolation System, Promega, Madison, Wis.), according to manufacturer's instructions with a slight modification for serum or plasma samples: to each 100 μl serum or plasma were directly added 175 μl of SV RNA lysis buffer (only fresh or once frozen-thawed serum was used).

Detection of hTR, hTERT, TEP 1 and the rRNA (reference RNA) by the means of reverse transcriptase PCR (RT PCR):

The Qiagen One Step RT-PCR kit (Qiagen, Basel, Switzerland) was used to detect the presence and the quantity of hTR and GAPDH RNA in serum. 1 mg of tumor RNA was used in a 25 μl RT-PCR reaction mixture containing 400 μM of each dNTP, omniscript™ reverse transcriptase, sensiscript™ reverse transcriptase, hot-start Taq™ DNA polymerase and 0.15 μM primers i.e. for hTR(sense)GAAGGGCG-TAGGCGCCGTGCTTTTC (SEQ ID NO: 1) and(antisense) GTTTGCTCTAGAATGAACGGTGGAAGG (SEQ ID NO: 2). The RT-PCT conditions of the mixture were an initial incubation at 50° C. for 30 min followed by a 95° C. incubation for 15 min to activate the HotstarTaq™ DNA Polymerase, then 50 cycles at 94° C. (30 sec), 65° C. (1 min), 72° C. (1 min) and a 10 minute final extension at 72° C.

RT-PCR conditions were identical for the detection of hTERT, except that 0.3 μM primer concentration was used, primers being (sense) TGACACCTCACCTCACCCAC (SEQ ID NO: 3) and (antisense) CACTGTCTTCCGCAAGTTCAC (SEQ ID NO: 4).

The same conditions were used for the detection of TEP 1, primers being (sense) CACCTGCGACGATATTTCT (SEQ ID NO: 5) and (antisense) CGAGGGTTGTACTTAGCCA (SEQ ID NO: 6) (primer concentration=0,3 μM).

In the case of control RNA, the same conditions as for hTR were used with a primer concentration of 0,3 μM; the sequences of the GAPDH primers being(sense)GGAGTCAACGGATTTGGTCGTAT (SEQ ID NO: 7) and (antisense) AGCCTTCTCCATGGTGGTGAAGAC (SEQ ID NO: 8).

All base sequences mentioned here above as primer examples are known and may as such be consulted on the web site of the Genome Database.

PCR amplification yielded products of 111 bp for hTR, 95 bp for hTERT, 150 bp for TEP1 and 306 bp for GAPDH RNA. Products were run at 55° C. on Elchrom Scientific S-50 gels (Elchrom Scientific, Cham, Switzerland), stained with SYBR-gold (Molecular Probes, Eugene, Oreg.-USA) for 45 min and destained twice in a darkroom for 30 min with deionized water at room temperature.

Results:

Using the technique described above, the result is that it is effectively possible to detect a PCR product of the reference RNA in all samples analysed. Quantitatively, all samples are similar whether they come from the serum of a healthy person or of a sick person.

In the case of the amplification product obtained with the specific amplimers for hTR, only the RNA of patients suffering of breast cancer was positive in 25% of the cases. The other RNA component of the enzyme hTERT was detected in 22% of the sera of patients. TEP 1 was detected in 20% of the cases. All together, the combination of the three markers has allowed th detection of breast cancer in 55% of the cases; this rate is clearly higher than the rate one may obtain with techniques known up to now that rarely exceed 30%. Moreover the RNA components of the telomerase are absent in the sera of the controls (healthy persons). 

1. A method of detecting human telomerase reverse transcriptase enzyme (hTERT) RNA extracted from plasma or serum from a human comprising: a) extracting a sample comprising extracellular RNA from plasma or serum of a human; b) amplifying said extracellular RNA for hTERT RNA to obtain an amplified product; c) detecting whether hTERT is present in the amplified product; and d) quantifying the amount of extracellular hTERT RNA in said sample, to thereby detect said hTERT RNA.
 2. The method of claim 1, wherein said amplifying comprises reverse transcribing and amplifying the hTERT RNA in vivo.
 3. The method of claim 2, wherein amplification is performed by polymerase chain reaction.
 4. A method for detecting telomerase associated protein (TEP1) RNA in plasma or serum of a human comprising the steps of: a) extracting a sample comprising extracellular RNA from plasma or serum of a human; b) amplifying said extracellular RNA for TEP1 RNA; c) detecting whether TEP1 is present in said sample; and d) quantifying the amount of extracellular TEP1 RNA in said sample, to thereby detect said TEP1 RNA.
 5. The method of claim 4, wherein said amplifying comprises reverse transcribing and amplifying the TEP1 RNA.
 6. The method of claim 4, wherein amplification is performed by polymerase chain reaction.
 7. A method for diagnosing or monitoring cancer in a patient, comprising: extracting extracellular RNA from plasma or serum of the blood from said patient; amplifying said extracellular RNA for an RNA component, wherein said RNA component is human telomerase reverse transcriptase enzyme (hTERT) RNA to detect whether said plasma or serum of the blood from said patient contains said RNA component; quantifying the amount of said RNA component, if present, in said plasma or serum; and comparing the amount of said RNA component in the plasma or serum of the blood from said patient to the amount of said RNA component in the plasma or serum of the blood a healthy person to diagnose or monitor cancer in said patient.
 8. The method according to claim 7, further comprising detecting and quantifying the amount of GADPH RNA in the plasma or serum of the blood of the patient.
 9. The method according to claim 7, further comprising detecting and quantifying the amount of human telomerase RNA (hTR) in the plasma or serum of the blood of the patient.
 10. A method for diagnosing or monitoring cancer in a patient, comprising: determining the level of extracellular human telomerase reverse transcriptase enzyme (hTERT) RNA in the plasma or serum of the blood of a patient having or suspected of having a malignancy, and comparing the level of said extracellular hTERT RNA in the plasma or serum of the blood of said patient to the level of extracellular hTERT RNA in the plasma or serum of the blood of a healthy person to thereby diagnose or monitor cancer in said patient.
 11. The method according to claim 10, further comprising detecting and quantifying the amount of GADPH RNA in the plasma or serum of the blood of the patient.
 12. The method according to claim 10, further comprising comparing the extracellular RNA levels of human telomerase RNA (hTR) from the plasma or serum of the blood of the patient having or suspected of having a malignancy to the extracellular RNA levels of hTR from the plasma or serum of the blood of a healthy person to diagnose or monitor cancer in said patient.
 13. A method for diagnosing or monitoring cancer in a patient, comprising: extracting extracellular RNA from plasma or serum of the blood from said patient; amplifying said extracellular RNA for an RNA component, wherein said RNA component is telomerase associated protein (TEP1) RNA to detect whether said plasma or serum of the blood from said patient contains said RNA component; quantifying the amount of said RNA component, if present, in said plasma or serum; and comparing the amount of said RNA component in the plasma or serum of the blood from said patient to the amount of said RNA component in the plasma or serum of the blood a healthy person to diagnose or monitor cancer in said patient.
 14. A method for diagnosing or monitoring cancer in a patient, comprising: extracting extracellular RNA from plasma or serum of the blood from said patient; amplifying said extracellular RNA for RNA components, wherein said RNA components are human telomerase reverse transcriptase enzyme (hTERT) RNA and telomerase associated protein (TEP1) RNA to detect whether said plasma or serum of the blood from said patient contains said RNA component; quantifying the amount of said RNA components, if present, in said plasma or serum; and comparing the amount of said RNA components in the plasma or serum of the blood from said patient to the amount of said RNA components in the plasma or serum of the blood a healthy person to diagnose or monitor cancer in said patient.
 15. The method according to claim 13 or 14, further comprising detecting and quantifying the amount of GADPH RNA in the plasma or serum of the blood of the patient.
 16. The method according to claim 13 or 14, further comprising detecting and quantifying the amount of human telomerase RNA (hTR) in the plasma or serum of the blood of the patient.
 17. A method for diagnosing or monitoring cancer in a patient, comprising: determining the level of extracellular telomerase associated protein (TEP1) RNA in the plasma or serum of the blood of a patient having or suspected of having a malignancy, and comparing the level of said extracellular TEP1 RNA in the plasma or serum of the blood of said patient to the level of extracellular TEP1 RNA in the plasma or serum of the blood of a healthy person to thereby diagnose or monitor cancer in said patient.
 18. A method for diagnosing or monitoring cancer in a patient, comprising: determining the level of extracellular RNAs in the plasma or serum of the blood of a patient having or suspected of having a malignancy, wherein said extracellular RNAs are human telomerase reverse transcriptase enzyme (hTERT) RNA and telomerase associated protein (TEP1) RNA, and comparing the level of said extracellular hTERT and TEP1 RNAs in the plasma or serum of the blood of said patient to the level of extracellular hTERT and TEP1 RNA in the plasma or serum of the blood of a healthy person to thereby diagnose or monitor cancer in said patient.
 19. The method according to claim 17 or 18, further comprising detecting and quantifying the amount of GADPH RNA in the plasma or serum of the blood of the patient.
 20. The method according to claim 17 or 18, further comprising comparing the extracellular RNA levels of human telomerase RNA (hTR) from the plasma or serum of the blood of the patient having or suspected of having a malignancy to the extracellular RNA levels of hTR from the plasma or serum of the blood of a healthy person to diagnose or monitor cancer in said patient. 